Project Summary We propose a design-directed engineering solution to address challenges in live, single-cell investigations, thus enabling new opportunities for biomedical research. Understanding the dynamics of individual cells is crucial to unraveling the idiosyncratic behaviors involved in complex disease states, yet traditional techniques fail to capture these heterogeneities in real-time. These live, single-cell investigations are particularly necessary for infections by persistent intracellular bacteria such as Staphylococcus aureus (SA) that can survive within host cells for prolonged periods, leading to relapsing disease that resists treatment. To investigate the broad biochemical repertoire involved in such host-pathogen interactions, tools that enable multiplexed profiling of intracellular metabolites in tandem with dynamic transcriptomics are urgently needed. We propose the development of plasmonic nanoneedle substrates for Surface-Enhanced Raman Spectroscopy (SERS) that will be used to profile the intracellular miRNA and metabolite landscape of individual macrophages in real time during phagocytosis of SA. By fabricating silver and gold nanostructures onto silicon nanoneedles, we will create a plasmonic substrate capable of interrogating live cells. Raman imaging of substrates functionalized with chemical probes will be used for real-time characterization of SA phenotype switching, quantification of local antibiotic concentration, determination of bacterial alterations to intracellular pH, and investigation of the interplay of reactive oxygen and nitrogen species. Furthermore, we will implement a novel kinetically-regulated miRNA probe architecture specifically tailored to real-time sequence-specific monitoring of miRNA expression. The feature-rich SERS spectra of Raman dyes conjugated to such oligonucleotide beacons decorating the nanoneedle surface will enable sensitive detection of specific miRNA sequences within SA-infected macrophages at high multiplexing capacity, enabling us to investigate the idiosyncratic differences between those macrophages which effectively destroy SA immediately upon phagocytosis and those which only destroy SA after TNF-? stimulation, or not at all. In addition to the technical training provided over the course of this fellowship, the Fellow will be provided with professional development opportunities to expand his expertise in lab management, grant writing, scientific communication, and mentorship that will prepare him for the independent academic career he is dedicated to. The research proposed here will produce a greater understanding of the temporal host-cell response to dynamic infection events. Real-time observations of miRNA expression may identify candidates for siRNA-mediated immunotherapy and will provide an essential tool for future characterizations of transcriptional responses to various stimuli. Upon completion of our aims, researchers will be capable of highly detailed explorations into the cellular repertoire of chemical, transcriptional, and phenotypic responses in a broad range of contexts.